Composition for treating cancer cells and preparation method for the same

ABSTRACT

A composition for treating cancer cells and a preparation method therefor are provided. The novel flavonoid compounds are obtained from natural plants, and more particularly the compounds have a cytotoxicity on cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of and claims the benefit of priority from U.S.patent application Ser. No. 12/465,925, filed May 14, 2009 now U.S. Pat.No. 7,670,630, which in turn is a divisional and claims the benefit ofU.S. patent application Ser. No. 11/471,295, filed Jun. 20, 2006, nowU.S. Pat. No. 7,550,160, the full disclosures of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a composition for treating cancer cellsand a preparation method therefor, and more particularly to thecomposition comprising extracts of Thelypteris torresiana and thepreparation method therefor.

BACKGROUND OF THE INVENTION

Recently many extracts and their derivatives oriented from naturalplants, such as a vincristinem and a vinbalstine, a camptothecin, ataxol and its derivatives, a palitaxel and a docetaxel, have been widelyused in clinical chemical therapy of a malignant tumor. Therefore, theeffect of the extracts from natural plants has been a burgeoningresearch in the field of new drug development.

Flavonoids are widely distributed in plants fulfilling many functionsincluding producing yellow or red/blue pigmentation in flowers andprotection from attack by microbes and insects. The widespreaddistribution of flavonoids, their variety and their relatively lowtoxicity compared to other active plant compounds (for instancealkaloids) mean that many animals, including humans, ingest significantquantities in their diet. Flavonoids have been referred to as “nature'sbiological response modifiers” because of strong experimental evidenceof their ability to modify the body's reaction to allergens, viruses,and carcinogens. They show anti-allergic, anti-inflammatory,anti-microbial and anti-cancer activity. In addition, flavonoids act aspowerful antioxidants, providing remarkable protection against oxidativeand free radical damage.

As a result, consumers and food manufacturers have become increasinglyinterested in flavonoids for their medicinal properties, especiallytheir potentially beneficial role in the prevention of cancers andcardiovascular disease. The beneficial effects of fruit, vegetables, andtea or even red wine have been attributed to flavonoid compounds ratherthan to known nutrients and vitamins. These beneficial effects awaitfurther clinical trials in humans.

In view of the above, the inventors of the present invention discoverthat the crude extracts of Thelypteris torresiana, only produced inTaiwan, have cytotoxic activities to the NUGC and HONE-1 cell linessupported by the experimental data through screening several dozens ofnatural crude extracts based on the inventors' experience in studyingthe effect of the extracts from natural plants for a long time. To studyin advance, it is discovered that the unique novel flavonoid compoundstherein have strong cytotoxic activities to human cancer cells,including liver cancer cells (Hep G2, and Hep 3B), breast cancer cells(MCF-7, MDA-MB-231), and lung cancer cells (A549).

SUMMARY OF THE INVENTION

The present invention provides a composition for treating cancer cellsand a preparation method therefor, wherein not only the novel flavonoidcompounds are obtained from natural plants but also the biologicalactivity thereof is capable of treating cancer cells.

In accordance with one aspect of the present invention, a compositionfor treating a cancer cell is provided. The composition comprises aflavonoid compound represented by a formula (I):

B ring is 4-oxo-cyclohexa-2,5-dienyl group, and the R₁, R₂, R₃ and R₄are respective ones selected from the group consisting of H, OH,C₁₋₁₂-alkyl, C₁₋₁₂-alkoxy, and sugar groups.

According to the present invention, the flavonoid compound is extractedfrom Pteridophyta.

According to the present invention, the Pteridophyta is Thelypteristorresiana.

In accordance with another aspect of the present invention, acomposition for treating a cancer cell is provided. The compositioncomprises a flavonoid compound represented by a formula (II):

B ring is 4-oxo-cyclohexa-2-enyl group, and the R₁, R₂, R₃, R₄, and R₅are respective ones selected from the group consisting of H, OH,C₁₋₁₂-alkyl, C₁₋₁₂-alkoxy, and sugar groups.

According to the present invention, the flavonoid compound is extractedfrom Pteridophyta.

According to the present invention, the Pteridophyta is Thelypteristorresiana.

In accordance with a further aspect of the present invention, acomposition for treating a cancer cell is provided. The compositioncomprises a flavonoid compound represented by a formula (III):

B ring is cyclohexa-2,5-dienol group, and the R₁, R₂, R₃, and R₄ arerespective ones selected from the group consisting of H, OH,C₁₋₁₂-alkyl, C₁₋₁₂-alkoxy, and sugar groups.

According to the present invention, the flavonoid compound is extractedfrom Pteridophyta.

According to the present invention, the Pteridophyta is Thelypteristorresiana.

In accordance with further another aspect of the present invention, amethod for preparing one of the flavonoid compounds is provided. Thefollowing steps are comprised. First, a dried plant is provided. Then, afirst extract with a first organic solution from the dried T. torresianais obtained. Next, a second extract with a second organic solution fromthe first extract is obtained. Finally, the flavonoid compound isobtained by separating the second extract.

According to the above, the first organic solution is a methanol.

According to the above, the second organic solution is an ethyl acetate.

According to the above, the flavonoid compound is separated from thesecond extract by a chromatography.

The foregoing and other features and advantages of the present inventionwill be more clearly understood through the following descriptions withreference to the drawings, wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of the preparation method according to the presentinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically withreference to the following embodiments. It is to be noted that thefollowing descriptions of preferred embodiments of this invention arepresented herein for the purposes of illustration and description only;it is not intended to be exhaustive or to be limited to the precise formdisclosed.

The present invention provides a composition for treating cancer cells.The composition comprises a flavonoid compound, which is extracted fromT. torresiana. The preparation method is illustrated as follows.

Please refer to FIG. 1, which shows a flowchart of the preparationmethod according to the present invention. In the method of the presentinvention, 1.5 kilograms of total T. torresiana. Plant are gathered anddried, and then two liters of methanol are used to extract the dried T.torresiana. Plant for six times repeatedly to obtain a first extract.The first extract is formed as a brown sticky material afterdecompressing, condensation, and drying, and then the brown stickymaterial is separated by shaking in the buffer of the acetate and water.A second extract is obtained from the acetate layer throughdecompressing and condensation. Subsequently, a chromatography isperformed to fractionate the second extract using n-Hexane,n-Hexane/acetate (4:1), acetate, methanol, and water respectively in twoliters as a mobile phase in the resin of celite 545 to obtain fivefractions (T1-T5).

The fraction T4 is further processed repeatedly by chromatography in thesilica gel column using a chloroform/methanol as a mobile phrase. Bymeans of the preparation method, a huge amount of the flavonoidcompound, named as protoapigenone, can be obtained.

During the above separation process of T4 fraction, a pure compound,whose polarity is less than the protoapigenone, is obtained in a tinyamount using a chloroform/methanol as a mobile phrase. The pure compoundis also a flavonoid compound, named as5′,6′-dihydro-6′-methoxyprotoapigenone.

The fraction T3 is separated through a preparative high-performanceliquid chromatography using a methanol/water (50:50) as a mobile phrase.In the condition of 3.5 ml/min flow rate, a novel flavonoid compound isobtained, named as protoapigenin, at t_(R)=15.5 min. However, anothernovel compound is obtained, named as5,7-dihydro-2-(1-hydroxy-4,4-dimethoxy-cyclohexyl)-chromen-4-one, in thesame condition.

According to the preparation method of the present invention, there arefour novel flavonoid compounds obtained, and their respective propertiesare identified and described as follows.

With regard to the protoapigenone, it is a needle crystal with lightyellow color and its pseudo molecule ion peak m/z is 287.0553 [M+H]⁺with the molecular formula being C₁₅H₁₀O₆. In the ¹H NMR spectrumthereof, there is the signal that indicates the chemical shift of B ringis respectively δ7.23 and δ6.52 (which indicates two hydrogen, doublet,and the coupling constant being 10.2). The signal is presented in aspecial AA′XX′ system. Further, there is a signal of δ185.2 indicating aketo group at the 4′ position according to the ¹³C NMR spectrum.Furthermore, there is a set of retro-Diels-Alder split signals at C-ringin flavonoids, m/z [M−152]⁺ and [M−133]⁺, from analyzing the fragmentsin the mass spectrum.

As for the physical properties of the protoapigenone, this compound hasthe following properties: m.p. 180-181° C.; UV (MeOH): λ_(max) 325, 299,259, 249, 230, and 206 nm; IR: υ_(max) 3224, 2928, 1656, 1621, 1597,1349, and 1164 cm⁻¹; ¹H NMR (200 MHz, pyridine-d₅): δ=13.31 (1H, br s,OH-5), 7.23 (2H, d, J=10.2 Hz, H-2′, H-6′), 7.05 (1H, s, H-3), 6.72 (1H,d, J=2.2 Hz, H-8), 6.59 (1H, d, J=2.2 Hz, H-6), 6.52 (2H, d, J=10.2 Hz,H-3′, H-5′). EI-MS m/z (rel. int.): 286 [M]⁺ (100), 270 (39), 242 (18),229 (31), 153 (33), 134 (12); HR-ESI-MS: m/z 287.0553 [M+H]⁺ (Calculatedfor C₁₅H₁₁O₆, 287.0550).

With regard to 5′,6′-dihydro-6′-methoxyprotoapigenone, it is a lightyellow solid and its pseudo molecule ion peak m/z is 319.0814 [M+H]⁺with the molecular formula being C₁₆H₁₅O₇. Its ultraviolet and infraredspectrum is similar to the protoapigenone, indicating that the compoundhas the typical skeleton of flavonoid compounds. The5′,6′-dihydro-6′-methoxyprotoapigenone is derivated from the reductionof the double bond between 5′ and 6′ positions, and the 6′ position ofthe protoapigenone is replaced with a methoxyl group based on theanalysis of 1D and 2D NMR spectrum signals. A retro-Diels-Alder (RDA)signal, m/z [M−165]⁺, of C-ring in flavonoids is obtained by analyzingthe fragments in the mass spectrum. This compound has the followingproperties: m.p. 162-163° C.; UV (MeOH): λ_(max) 326, 297, 254, 232, 212nm; IR: ν_(max) 3350, 2940, 1658, 1624, 1583, 1352, 1165 cm⁻¹; ¹H NMR(400 MHz, pyridine-d₅) δ=13.42 (1H, br s, OH-5), 7.13 (1H, d, J=10.0 Hz,H-2′), 7.12 (1H, s, H-3), 6.77 (1H, d, J=2.0 Hz, H-8), 6.75 (1H, d,J=2.0 Hz, H-6), 6.38 (1H, d, J=10.0 Hz, H-3′), 4.33 (1H, dd, J=9.2, 4.4Hz, H-6′), 3.31 (3H, s, OCH₃), 3.21 (1H, dd, J=16.0, 9.2 Hz, H-5′ a),3.15 (1H, dd, J=16.0, 4.4 Hz, H-5′ b); EI-MS m/z (rel. int.); 318 [M]⁺(19), 260 (100), 232 (39), 218 (19), 204 (36), 203 (35), 153 (7);HR-ESI-MS: m/z 319.0814 [M+H]⁺ (Calculated for C₁₅H₁₁O₆, 319.0812).

With regard to protoapigenin, it is a light yellow solid and its falsemolecule ion peak m/z is 289.0714 [M+H]⁺ with the molecular formulabeing C₁₅H₁₂O₆. Its ¹³C NMR spectrum signal is similar to that of theprotoapigenone, and the apparent difference is the vanished signal ofδ185.2 and the additional signal of δ61.9, which indicates that theketonyl group at the 4′ position of the protoapigenone is reduced to ahydroxyl group. Further, its ¹H NMR spectrum and 2D NMR signals are alsoin support of the above result. Therefore, a set of retro-Diels-Alder(RDA) signals, m/z [M−153]⁺ and [M−136]⁺, at C-ring in flavonoids areobtained by analyzing the fragments in the mass spectrum. This compoundhas the following properties: m.p. 192-193° C.; UV (MeOH): λ_(max) 323,299, 258, 249, 2320 nm; IR: ν_(max) 3356, 2924, 1656, 1610, 1553, 1368,1073 cm⁻¹; ¹H NMR (400 MHz, pyridine-d₅) δ=13.52 (1H, br s, OH-5), 6.90(1H, s, H-3), 6.71 (1H, d, J=2.4 Hz, H-8), 6.54 (1H, d, J=2.4 Hz, H-6),6.53 (2H, dd, J=10.2, 1.6 Hz, H-2′, H-6′), 6.36 (2H, dd, J=10.2, 3.2 Hz,H-3′, H-5′), 4.78 (1H, m, H-4′); EI-MS m/z (rel. int.): 288 [M]⁺ (35),270 (28), 194 (20), 153 (82), 124 (43), 121 (76), 111 (60), 69 (100);HR-ESI-MS: m/z 289.0714 [M+H]⁺ (Calculated for C₁₅H₁₁O₆, 289.0712).

With regard to the5,7-dihydroxy-2-(1-hydroxy-4,4-dimethoxy-cyclohexyl)-chromen-4-one, itis a light yellow solid and its pseudo molecule ion peak m/z is 359.1106[M+H]⁺ with the molecular formula being C₁₇H₂₀O₇Na. The 1D and 2D NMRspectrums indicate that this compound has the similar skeleton offlavonoids, and only the alkenyl groups on 2′, 3′, 5′, and 6′ positionsat B-ring is reduced to alkyl groups. It is also found that the signalsat 3′ and 5′ positions have a tendency towards a high magnetic field.Therefore, it is concluded that there are two methoxyl groups at 4′position. This compound has the following properties: ¹H NMR (400 MHz,pyridine-d₅) δ=13.57 (1H, br s, OH-5), 6.97 (1H, s, H-3), 6.75 (1H, d,J=2.0 Hz, H-8), 6.69 (1H, d, J=2.0 Hz, H-6), 3.22 and 3.19 (each 3H, s,OCH₃, exchangeable), 2.33 (2H, td, J=13.6, 3.6 Hz, H-2a, 6a), 2.21 (2H,td, J=13.6, 3.6 Hz, H-3a, 5a), 2.07 (2H, br.d, J=13.6, H-3b, 5b), 1.99(2H, br.d, J=12.6, H-2b, 6b); HR-ESI-MS: m/z 359.1106 [M+H]⁺ (calculatedfor C₁₅H₁₁O₆Na, 359.1107).

Furthermore, the above four novel flavonoid compounds of the is presentinvention are tested for their biological activities. There are fivehuman cancer cell lines for a biological activity testing, including twohuman breast cancer cell lines (MCF-7 and MDA-MB-231), two human livercancer cell lines (Hep G2 and Hep 3B), and one human lung cancer cellline (A549). Besides, there is one human breast epidermal cell (MCF-10A)as a normal control. Human cancer cells are from American Type CultureCollection, and incubated in the RPMI-1640 suspension with the additivesof the 10% (v/v) fetal calf serum, 100 U/ml penicillin, and 100 μg/mlstreptomycin under the condition of 37° C., 5% carbon dioxide, and 95%fresh air. Subsequently, the MCF-10A cell line is incubated in 50%Dulbecco's modified Eagle's buffer and the 50% Ham's F-12 buffer with 10μg/ml cow trypsin, 20 ng/ml EGF, 100 ng/ml cholera enterotoxin, 0.5μg/ml hydrocortisone, and 10% fetal calf serum.

In the present invention, the cytotoxic testing experiment is analyzedin the method of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) known in the prior art. The respective five cancercell lines and the normal cell line (MCF-10A) are incubated within themicroplate with 96 wells in the density of 5,000-10,000 cells/well. Thenext day the detected compounds (A-D) and Doxorubicin (compound E) areused to treat the cells. Subsequently, the treated cells are dissolvedin DMSO according to the MTT method. Finally, the testing result isobtained by detecting the absorption at 550 nm to analyze the cytotoxicactivities of respective compounds. The testing result is shown inTable 1. The value of IC₅₀ represents the compound concentration forinhibiting 50% cells growth, and is the Doxorubicin (compound E) havinga cytotoxicity on cancer cells is served as a positive control.

IC₅₀ (μg/mL)^(a) MDA-MB- Compound Hep G2 Hep 3B MCF-7 A549 231 MCF-10A A1.60 ± 0.33 0.23 ± 0.01 0.78 ± 0.02 3.88 ± 0.02 0.27 ± 0.02 7.9 ± 1.3 B5.88 ± 0.47 1.74 ± 0.08 5.92 ± 0.29 13.30 ± 0.23  1.30 ± 0.10 *^(c) C1.60 ± 0.33 20.00 ± 0.58  — 18.84 ± 0.60  — * D —^(b) — — — — * E 0.15 ±0.00 0.26 ± 0.06 0.32 ± 0.08 0.36 ± 0.04 0.31 ± 0.02 * Compound A:Protoapigenone Compound B: 5′,6′-Dihydro-6′-methoxy-protoapigenoneCompound C: Protoapigenin Compound D:5,7-dihydroxy-2-(1-hydroxy-4,4-dimethoxy-cyclohexyl)-chromen-4-oneCompound E: Doxorubicin ^(a)The data is represented as the average value± standard deviation (n = 2) ^(b)Without the cytotoxical activity in theconcentration of 20 μg/ml ^(c)The test is not performed

As shown in Table 1, the protoapigenone,5′,6′-dihydro-6′-methoxy-protoapigenone, and protoapigenin are proved tohave good cytotoxic activities on cancer cells.

According to the past literatures, Peterson J. et al (Nutrition Research1998) disclosed that the general flavonoid compounds only have a weakcytotoxity. However, 1-oxygenated 4-oxo-cyclohexa-2,5-dienyl group,4-oxo-cyclohexa-2-enyl group, cyclohexa-2,5-dienol or cyclohexanel groupat B-ring in the flavonoid compounds of the present invention areextracted and identified. The biological activities thereof confirm thatthe novel compounds disclosed in the present invention have goodcytotoxicity on five human cancer cell lines. Therefore, these kind ofspecial flavonoid compounds indicate their unique biological activity,and more particularly, the protoapigenone of the present invention hasweak cytotoxicity to normal human breast epidermal cells, which impliesone compound with the 1-oxygenated 4-oxo-cyclohexa-2,5-dienyl group atB-ring is a selectivity marker for cancer cells. Therefore, the aboveunique biological activity is apparently different from the generalflavonoid compounds.

According to the above, a composition for treating cancer cells and itspreparation method of the present invention not only obtains the novelflavonoid compounds from natural T. torresiana but more particularly thebiological activity thereof with a cytotoxicity.

While the invention has been described in terms of what is presentlyconsidered to be the most practical and preferred embodiments, it is tobe understood that the invention needs not be limited to the disclosedembodiments. On the contrary, it is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the appended claims which are to be accorded with the broadestinterpretation so as to encompass all such modifications and similarstructures.

1. A composition for treating cancer cells, comprising an effectiveamount of an isolated flavonoid compound of a formula (III):

wherein B ring is cyclohexa-2,5-dienol group, and the R₁, R₂, R₃, and R₄are irrespective ones selected from the group consisting of H, OH,C₁₋₁₂-alkyl, C₁₋₁₂-alkoxy, and sugar groups.
 2. The composition of claim1, wherein the isolated flavonoid compound is extracted fromPteridophyta.
 3. The composition of claim 2, wherein the Pteridophyta isThelypteris torresiana.